Production of nucleotide anhydrides

ABSTRACT

NUCLEOTIDE ANHYDRIDES CAN BE SYNTHESIZED, IN EACH CASE, BY A SINGLE-STEP PROCESS BY CAUSING A NUCLEOSIDE-5&#39;&#39;-MONOPHOSPHATE AND DIPHENYL PHOSPHOROCHLORIDATE OR TETRAPHENYL PYROPHOSPHATE TO REACT IN A SUITABLE SOLVENT AND IN THE PRESENCE OF AN ALKYLAMINE AND THEN ADDING TO THE RESULTING PROCESS MATERIALS ALKYLAMMONIUM SALT OF AN ACID DISSOLVED IN PYRIDINE OR ONE OF ITS DERIVATIVES.

United States Patent PRODUCTION OF NUCLEOTIDE ANHYDRIDES KiminoriTamura, Manami Morozumi, Yutaka Noda, Morio Suzuki, and Hiroshi Yoshino,Choshi-shi, Japan, assignors to Yamasa Shoyu Kabushiki Kaisha, Araoi,Choshi-shi, Chiba-ken, Japan No Drawing. Filed June 1, 1970, Ser. No.42,579 Claims priority, application Japan, June 12, 1969, 44/ 45,786Int. Cl. C07d- 51/50 US. Cl. 260-211.5 R 4 Claims ABSTRACT OF THEDISCLOSURE Nucleotide anhydrides can be synthesized, in each case, by asingle-step process by causing a nucleoside-S'-monophosphate anddiphenyl phosphorochloridate or tetraphenyl pyrophosphate to react in asuitable solvent and in the presence of an alkylamine and then adding tothe resulting process materials alkylammoniurn salt of an acid dissolvedin pyridine or one of its derivatives.

BACKGROUND OF THE INVENTION This invention relates generally tonucleotide anhydrides and production thereof and more particularly to anew and advanced process for producing nucleotide anhydrides by aone-step synthesis in a short time and in an economical manner.

The term nucleotide anhydride is herein used to designate an anhydrideof nucleoside-5'-monophosphate and an acid whose acidity is weaker thandiphenylphosphate. The anhydrides can be represented by the followinggeneral formula and consist of the compounds indicated in Table 1.

These nucleotide anhydrides are very important substances for themetabolism within living organisms, and

pharmaceutical and biochemical demand thereof is recently increasing.

For example, CDP-choline is effective for treatment of external headinjuries, coma due to external injury 3,701,772 Patented Oct. 31, 1972to the brain, etc. Trisodium inosine-5'-triphosphate (ITP-Na andtrisodium thymidine-S'-triphosphate (TIP-Na are present within musclesand are valuable reagents for research on metabolism within livingorganisms. Adenosine triphosphate (ATP) is used for the circulation,particularly peripheral vascular disturbances, edema, beri-beri, andmuscular fatigue and for the treatment of myositis (myitis, sarcitis),myasthenia, rheumatosis, arthritis, neuralgia, and other similarailments.

Flavine adenine dinucleotide (FAD) is used as a medicine in thetreatment of hepatic disturbances, trophopathy in pregnant women andlactating (breast-feeding) women, and toxicosis (toxinosis, toxipathy)such as alcohol damage and nicotinism. In addition, disodiumuridine-S'-diphosphoglucose (UDPG-Na is of physiological value, andCDP-ethanolamine can be used as one kind of nucleotide coenzyme.Moreover, uridylyl sulfate, adenosine-S-2,4-dinitrophenyl phosphate,adenyl-5'-yl carbobenzy oxyglycine, and other nucleotide anhydrides arehighly promising for future development and application as medicines andbiochemical reagents.

Among various methods so far established for synthesizing thesenucleotide anhydrides, the anion-exchange method is considered atpresent to be the most advantageous. The anion-exchange method comprisesthe following two steps. The first step is reaction of a nucleoside-5'-monophosphate with tetraphenyl pyrophosphate or diphenylphosphorochloridate in the presence of a tertiary base in dioxane or amixture of dioxane and dimethyl formamide.

The reaction product is P -nucleoside-5-P -diphenyl pyrophosphate. Thesecond step is reaction of purified P -nucleoside-5'-P -diphenylpyrophosphate with an acid (whose acidity is weaker than diphenylphosphate) to produce a nucleotide anhydride. The second reaction isbased on the transition elfect of pyridine. Variousnucleoside-S-monophosphates and acids can be combined arbitrarily by theanion exchange method to prepare various nucleotide anhydrides.

In this case, the purification of the intermediate product obtained bythe first reaction is accomplished by concentrating the reaction mixtureunder reduced pressure to remove the solvent, adding ethyl ether to theresulting residue to precipitate the compound, letting the processmaterial stand at 0 C. for 30 to 60 minutes, and removing the ether bydecantation. However, since this intermediate material is unstable whenexposed to moisture and heat, it is necessary to carry out theconcentration under an anhydrous condition, at a low temperature, and ina high vacuum. Furthermore, an extremely large quantity of ethyl etheris required for precipitation, giving rise to great danger for a largescale operation. Thus the method is industrially disadvantageous.Moreover, in the process for isolation of the intermediate material, itssignificant loss is unavoidable.

Furthermore, water or ethyl ether admixed with the intermediate materialobtained by precipitation markedly inhibits the second reaction andlowers the yield. Accordingly, it is disadvantageously necessary toresort to a process step of dissolving the precipitate in dioxane andconcentrating the resulting solution under reduced pressure to romvethese liquids by azeotropy.

Thus, the above described known method requires two stages of reaction,and the operation for isolating the intermediate material, moreover, iscomplicated and requires a long time and complicated productionequipment. Furthermore, a large quantity of the solvent is necessary,and a considerable amount of the intermediate material is lost.

On account of these difiiculties this method is not suitable forindustrial production.

3 SUMMARY OF THE INVENTION It is an object of the present invention toovercome the above described difficulties by utilizing certain findingswe have made and thereby to provide a new advanced process forsynthesizing nucleotide anhydrides whereby the reaction can be completedin a short time in one step by a simple operation without isolation ofthe intermediate materials.

We have found that a nucleotide anhydride can be synthesized in a singleoperation by causing a nucleoside-'- monophosphate and diphenylphosphochloridate or tetraphenyl pyrophosphate to react in a suitablesolvent and in the presence of an amine and then adding thereto an acidwhich .is weaker than diphenyl phosphate dissolved in pyridine or one ofits derivatives.

According to the present invention, briefly summarized, there isprovided a process for synthesizing nucleotide anhydrides whereindiphenyl phosphorochloridate or tetraphenyl pyrophosphate is added to analkyl-ammonium saltof a nucleoside-S'-monophosphate in dioxane, N,N-dimethylformamide or N,N-dirnethylac'etamide and in the presence of analkylamine, and, to the resulting process materials, a solution inpyridine of an alkalammonium salt of an acid to be condensed with the5-phosphate part of the desired nucleotide.

The nature, details, and utility of the invention will be more clearlyapparent from the following detiled description beginning with generalconsiderations and concluding with specificexamples of practiceillustrating preferred embodiments of the invention.

DETAILED DESCRIPTION A nucleoside-S'-monophosphate is generally used inthe form of an alkylammonium salt in order to facilitate dissolutionthereof in a reaction solvent. Particularly in consideration ofsolubility in the solvent, use o-f a salt of a trialkylamine orquarternary ammonium hydroxide (for example, tri-n-buthylamine,tri-n-octylamine, methyl-trin-butylammonium hydroxide, ormethyl-tri-n-octylammonium hydroxide) is recommended.

Depending on the molar ratio between the nucleoside- 5'-monophosphateand alkylamine base in condensation, the mono (alkylarnmonium) salt andthe di alkylammonium) salt may be obtained.

While either kind of salt can be used, the mono (alkylammonium) salt isprincipally used because of its reactivity.

In the present invention, the following nucleotides can be employed asone of starting materials:

Natural nucleotides such as 5'-cytidylic acid, 5'-deoxyadenylic acid,5'-deoxyinosinic acid, 5'-deoxyguanylic acid, 5'-deoxycytidylic acid,5'-thymidylic acid, and adenosine (2,3'-cyclic phosphate) 5'-phosphateand unnaturalnucleotides such as 2-methyl-thio-5-inosinic acid etc.

Any (reaction) solvent, whichv dissolves alkylammonium. salts ofnuc1eoside-5'-phosphate and does not inhibit the reaction, can beemployed in this invention. Examples of. such reaction solvents We havefound to be suitable are N,N-dimethyl-.formamide, N,N-dimethylacetamide,and dioxane. While the minimum quantity of the solvent to be used is 5times (in moles) that of the nucleoside-5'- phosphate, a preferablerange is from to 50 times (in moles).

The above three solvents can be used not only alone, respectively, butalso as a mixture of two or three thereof. In addition, in order, toincrease the solubility of the reaction reagents, tetrahydrofuran,n-hexane, benzene, toluene, ethyl acetate, acetone, or chloroform can beadded to the solvent to an extent that the reaction is not inhibited.The limit to the quantity be added is 2 to 3 times by volume that of thereaction solvent, and equal volume or less is preferable.

Diphenyl phosphorochloridate or tetraphenylpyrophosphate should be usedso that its molar ratio to nucleoside- 5-phosphate is 1-2,preferentially 1-1.5.

Examples of alkylamine which is added as a reaction stabilizer aretrin-n-ethylarnine, tri-n-ethylamine, tri-noctylamine,N-methylpiperidine, and diethylaniline. The stabilizer should beemployed in an amount more than 0.5 mole, preferentially 1.2-2 moles,per one molar of the nucleoside-5'-phosphate.

After the reaction of nucleoside5-phosphate with diphenylphosphorochloridate or tetraphenylpyrophosphate, a desired aciddissolved in pyridine or its derivative is directly added to thereaction mixture, where the reaction forming nucleotide anhydride fromthe nucleoside- 5'-monophosphate and the acid can be observed. The addedacid should be a weaker acid than diphenyl phosphate. An acid whoseacidity is stronger than that of diphenyl phosphate failed to form anacid anhydride with nucleoside-5-phosphate.

Examples of an acid weaker than diphenyl phosphate are: Anhydroussulfuric acid, phosphates such as orthophosphate, pyrophosphate, andtriphosphate; sugar phosphates such as glucose-l-phosphate,mannose-l-phosphate, and galactose-l-phosphate; carboxylic acids such asacetic acid, and propionic acid, carbobenzyloxy amines; peptides;phenols such as 2,4-dinitrophenol; nucleosides; and acid constitutentsof coenzymes (for example, panthethin-4', 4'-bisphosphate, flavinemonoculeotide, phenyl phosphate, choline phosphate, and nicotinamidephosphate).

In consideration of the solubility of the acid in an organic solvent,the acid is ordinarily added in the form of a salt of an alkylamine as,for example, tri-n-butylamine or tri-n-octylamine.

The acid should be used in an amount of more than one mole,preferentially 1-2 moles, per one molar of the nucleoside-5-phosphate.

Substances Which dissolve in pyridine with difficulty, such as flavinenucleotide, is dissolved in another solvent such as a formamide beforemixing with pyridine.

It is essential to add pyridine or its derivative together with the acidsimultaneously: The reaction of the acid with P -nucleoside-5'-P-diphenyl' pyrophosphate is regarded as an anion exchange reaction Wherediphenyl phosphate (a strong acid) moiety of the latter compound isreplaced by the weak acid. Pyridine or its derivatives are essential forthis anion exchange reaction.

This is the reason why pyridine or its derivative should be addedtogether with the acid.

In addition to pyridine that is used satisfactorily, several pyridinederivatives such as picoline, lutidine, and cholidine can also be used.2-50 moles, particularly 5-30 moles, of pyridine or its derivative per 1mole of the nucleotide gives a good yield.

The reactions in the process according to the invention are generallycompleted in a relatively short time, although the time depends on thekind and quantity of the solvent used and the temperature.

P -nucleoside-5'-P -diphenyl pyrophosphate is usually synthesized almostimmediately after mixing nucleoside- 5-phosphate and diphenylphosphorochloridate or tetraphenyl pyrophosphate under the conditionsdescribed previously. Therefore, the acid dissolved in pyridine or itsderivative can be added to the reaction mixture almost immediately aftermixing nucleoside5'-phosphate and diphenylphosphorochloridate ortetraphenyl pyrophosphate. The anion exchange reaction is completed in afew minutes to a few hours, in a high yield, with formation of onlysmall amounts of by-products.

The anion exchange reaction can also be carried out a few minutes to afew hours after mixing nucleoside-5'- phosphate and diphenylphosphorochloridate or tetraphenyl pyrophate.

It is surprising that the nucleotide anhydride can be synthesizeddirectly in one vessel containing a complicated solvent system whereseveral materials are added successively. Furthermore, the synthesis ofthe nucleotide anhydride proceeds rapidly in a high yield, withformation of only small amounts of by-products.

The inventors discovered this surprising fact for the first time. Thepresent invention, based on this new discovery, thus establishes aneconomical process for synthesizing nucleotide anhydrides which is asuperior, as will be indicated hereinafter, to the known methodmentioned hereinbefore.

The nucleotide anhydrides produced by the process of the invention arepurified by a combination of processes such as ion exchange resin columnchromatography, and precipitation process with an organic solvent and/orother precipitants. The purified products were identified withrespective nucleotide anhydrides by means of paper chromatography withseveral solvents, and physical, chemical and enzymatic analyses.

In order to indicate still more fully the nature and utility of thepresent invention, the following specific examples of practiceconstituting preferred embodiments of the invention and results are setforth, it being understood that these examples are presented asillustrative only and that they are not intended to limit the scope ofthe invention.

Example 1.Trisodium cytidine-'-diphosphate (CDP a) To one mole of mono(methyl tri-in-butylammonium)- 5'-cytidylate, 1,500 mol. ofN,N-dimethylacetamide was added, and then 250 ml. of diphenylphosphorochloridate was added.

The mixture was agitated for 5 minutes to dissolve the materials. 500ml. of tri-n-butylamine and 2 l. of dioxane were then added to thebatch.

After stirring for 5 minutes, 2 l. of pyridine containing 2 moles ofmono (tri-n-butylammonium) phosphate was added to the batch.

After agitation, the mixture was left standing for one hour.

Upon completion of the reaction, the reaction mixture was concentratedin vacuo, added with 7 l. of water, then extracted with one litre" ofbenzene.

The water layer is diluted to 50 1., and applied to an anion exchangeresin Duolite A101D (Cl type, 101.) column. Cytidine-5'-diphosphate waseluted from the column with 0.1 N hydrochloride, and crystallized astrisodium salt (387 grammes (g.) (11.0 percent moisture)). Yield 73.5percent.

Molar ratio of the components is indicated in Table 2:

Example 2.Trisodium inosine-5'-triphosphate (ITP Na To 200 millimoles ofmono (tri-n-butylammoniurn) 5'-inosinate, 120 ml. of N,N-dimethylacetamide and mixture of 250 millimoles of tetraphenylpyrophosphate, 100ml. of tri-n-butylamine, and 600 ml. of dioxane were added successively.

After agitating 200 ml. of pyridine containing 400 millimoles of di(tri-n-butylammonium) pyrophosphate was then added to the batch.

The mixture was then left standing for 40 minutes synthesized inosine5'-triphosphate was isolated from the reaction mixture by means ofanion-exchange resin Duolite A101D (Cl type, 10 litres) columnchromatography, and obtained amorphous powder as trisodium salt. (107.9g. (10.3 percent moisture content)) Yield, 84.3 percent.

Molar ratio of the components is indicated in Table 3.

TABLE 3 Molar ratio Inosine Total P tfifiiiiiitifiifisiijiji i133 3135Example 3.-Trisodium thymidine-5'-triphosphate (TTP Na To 10 millimolesof mono (tri-n-butylammonium)-5'- thymidylate, 5 ml. ofN,N-dimethylformamide and mixture of 2.3 ml. ofdiphenylphosphorochloridate, 5 ml. of tri-n-butylamine, and 20 ml. ofdioxane were added successively, after agitating ml. of pyridinecontaining 20 millimoles of di (tri-n-butylammonium) pyrophosphate wasadded to the batch. Then, after 30 minutes, thymidine-5-triphosphate wasisolated from the reaction mixture as usual. The yield was 79.7 percent.

Example 4.-Disodium uridine-5'-diphosphoglucose (UDPG Na l0 millimolesof mono (tri-n-butylammonium) 5-uridylate was dissolved in 5 ml. ofN,N-dimethylformamine, and 5 m1. of dioxane, 6 ml. of tri-n-butylamine,and 2.3 ml. of diphenyl phosphorochloridate were added to the resultingsolution.

After 10 minutes 15 ml. of pyridine containing 12 millimoles of mono(tri-n-butylamr nonium) glucose-l-phosphate was added to the mixture.

After allowing to stand for two hours, the solvent was removed underreduced pressure.

The remaining substances were dissolved in water and applied to anionexchange resin Dowex 1 X8 (Cl column (100 ml.).Uridine-5-diphosphoglucose was eluted from the column as usual, andobtained amorphous powder as disodium salt (4.12 g.). Yield, 67.5percent.

Molar ratio of the components is indicated in Table 4.

In addition 98.6 percent of the preparation was dehydrogenized by theaction of UDPG dehydrogenase.

Example 5 .Adenosine-5-diphosphoglucose (ADPG) To one millimole of mono(methyl-tri-n-butylammonium) 5'-adenylate, 1 ml. of N,N-dimethy1acetamide, 0.22 ml., of dephenyl phosphorochloridate 1 ml. of dioxane,and 0.5 ml. of tri-n-butylamine were added successively.

After 30 minutes, 3 ml. of pyridine containing 1.5 millimoles of mono(tri-n-butylammoniurn) glucose-l-phosphate was added to the mixture.During one hour reaction, adenosine-5'-diphosphoglucose was synthesizedin a yield of 81.5 percent.

Example 6.Flavine-adenine-dinucleotide (FAD) One millimole of mono(tri-n-octylammonium) 5'- adenylate was dissolved in 1 ml. ofN,N-dimethyl acetamide, and to the resulting solution, 0.25 ml. ofdiphenyl phosphorochloridate 2 ml. of dioxane, and 0.5 ml. oftrin-butylamine were added.

Then 2 ml. of formamide containing 1.8 millimoles of a mono(methyl-tri-n-butylammonium) salt of flavine mononucleotide and 4 ml. ofpyridine was added to the mixture. During 1.5 hours reaction,flavine-adenine-dinucleotide was synthesized in a yield of 78.3 percent.

Example 7.CDP-ethanolamine Two millimoles of mono (methyl tri noctylammonium) 5-cytidylate was dissolved in 2 ml. of N,N-dirnethylformamide, and to resulting solution, 0.5 ml. of diphenylphosphorochloridate, 1 ml. of tri-n-butylamine, and 15 ml. of dioxanewere added, the resulting solution and then being mixed.

5 ml. of pyridine containing 3 millimoles of mono(methyl-tri-n-butylammonium) salt of o-phosphoethanolamine was thenadded to the mixture. During 30 minutes raction, CDP ethanolamine, wassynthesized in a yield of 52.7 percent.

Example 8.-Uridylyl sulfate 10 millimoles of mono (tri-n-octylammonium)5'-uridylate was dissolved in 20 ml. of dioxane, and to the resultingsolution, 2.5 ml. of diphenyl phosphorochloridate and 2.1 ml. oftriethylamine were added.

After the resulting batch was left standing for 5 minutes at roomtemperature, 20 millimoles of tri-n-butylammonium sulfate and 5 ml. ofpyridine were added to the mixture.

During one hours reaction uridylyl sulfate was synthe sized in a yieldof 40 percent.

Example 9.-Adenosine-5'-2,4-dinitrophenyl-phosphate To 1 millimole ofmono (methyl-tri-n-butylarnmonium) 5'-adenylate, 2 ml. of N,N-dimethylacetamide was added, and then 0.25 ml. of diphenyl phosphorochloridateand 0.6 ml. of tri-n-butylamine were added after allowing it to stand atroom temperature for one hour, 2 ml. of pyridine containing a solutionof 2 millimoles of 2,4- dinitrophenol was added to the mixture. During 2hours reaction at C., adenosine-S'-2,4-dinitrophenol phosphatesynthesized in a yield of 91 percent.

Example 10.Adenyl-'-yl-carbobenzyl oxyglycine To 1 millimole of mono(tri-n-octylammonium) 5- adenylate, 2 m1. of N,N-dimethy1 acetamide wasadded, after. agitation a mixture of 0.25 ml. of diphenylphosphorochloridate, 4 ml. of dioxane, and 0.5 ml. of tri-nbutylaminewere added to the solution, which was then thoroughly agitated fordissolution.

2 ml. of pyridine containing 250 mg. of carbobenzyloxyglycine was thenadded to the reaction mixture. During 3 hours reaction,adenyl-5'-yl-carbobenzyl oxyglycine was synthesized in a yield of 32percent.

What is claimed is:

1. A one-stage process for producing a nucleotide anhydride whichcomprises adding a member selected from the group consisting of diphenylphosphorochloridate and tetraphenyl pyrophosphate to an alkylammoniumsalt of a nucleoside-S'-monophosphate in a reaction solvent selectedfrom the group consisting of dioxane, dimethylformamide, anddimethylacetamide and in the presence of an alkylamine and adding to theresulting mixture an alkylammonium salt of an acid dissolved in pyridineor one of its derivatives whereby said anhydride is produced in a singlestage without isolation of an intermediate.

2. A process for producing a nucleotide anhydride anhydride as claimedin claim 1 in which the quantities of the principal substances usedtherein in terms of multiples of the quantity in moles of saidnucleoside-5-monophosphate are to 50 times of said dioxane,dimethylformamide or dimethylacetamide, l to 1.5 times of said diphenylphosphorochloridate, 2 to 10 times of said acid, and 5 to 30 times ofsaid pyridine solvent, and the process is carried out at a reactiontemperature of from 0 to 30 C.

3. A process for producing nucleotide anhydride as claimed in claim 2 inwhich:

said alkyl-ammonium salt of a nucleoside-S'-monophosphate is selectedfrom mono (methyl-tri-n-butylammonium)-5'- cytidylate,

mono (tri-n-butylammonium)5'-inosinate,

mono (tri-n-butylammonium) -5 -thymidylate,

mono (tri-n-butylammonium) -5 '-uridylate,

mono (methyl-tri-n-butylammonium) -5 adenylate,

mono (tri-n-octylammonium -5 '-adenylate,

mono methyktri-n-octylammonium) -5 cytidylate,

mono tri-n-octylammonium -5 -uridylate,

mono methyl-tri-n-butylammonium -5 adenylate, and

mono (tri-n-octyla'mmonium -5-adenylate;

and said alkyl-ammonium salt of an acid is selected from I mono(tri-n-butylammonium) phosphate,

di(tri-n-butylammonium)pyrophosphate,

mono (tri-n-butylammonium) glucose- 1- phosphate,

mono(methyl-tri-n-butylamrnonium)salt of flavine mononucleotide,

mono(methyl-tri-n-butylammonium)salt of flavine mononucleotide,

mono (methyl-tri-n-butylammonium) salt of o-phosphoethanolamine,

tri-n-butylammonium sulfate,

2,4-dinitrophenol, and

carbobenzyloxyglycine,

to produce, respectively, one of the compounds selected from trisodiumcytidine-5'-diphosphate (CD'P Na trisodium inosine-5'-triphosphate (ITPNa trisodium thymidine-S'-triphosphate (TIP Na disodium uridine 5'diphosphoglucose (UDPG N32), adenosine-5'-diphosphoglucose (ADPG),fiavine-adenine-dinucleotide (FAD), CDP-ethanolamine, uridylyl sulfate,adenosine-S'-2,4-dinitrophenylphosphate, and adenyl- '-yl-carbobenzyloxyglycine.

4. In a process for the preparation of a nucleotide anhydride by thesteps of reacting a nucleoside-5-monophosphate with a reagent selectedfrom a member of the group consisting of tetraphenyl pyrophosphate anddiphenyl phosphorochloridate in the presence of a tertiary base and in asolvent, isolating and purifying the resultant P -nucleoside-5'-P-dipheny1 pyrophosphate, and then reacting said P -nucleoside-5'-P-diphenyl pyrophosphate with an acid having a weaker acidity thandiphenyl phosphate to produce said anhydride, the improvement whichcomprises carrying out said preparation in a single stage withoutisolation and purification of said P -nucleoside- 5'-P -diphenylpyrophosphate by mixing an alkylammonium salt of saidnucleoside-5'-monophosphate with said reagent in a solvent which doesnot inhibit the reaction in the presence of an alkylamine and thenadding directly to the resultant mixture an alkylammonium salt of saidacid dissolved in pyridine or a pyridine derivative to form saidanhydride.

References Cited UNITED STATES PATENTS 3,089,869 5/1963 Mauvernay260--21l.5 R 3,299,043 1/ 1967 Schramm et al. 26021l.5 R 3,321,1465/1967 Moifatt 260-2115 R 3,321,463 5/ 1967 Moflatt 260211.5 R

LEWIS GOTTS, Primary Examiner J. R. BROWN, Assistant Examiner US. Cl.X.R. 260-9'99

